Journal of Encapsulation and Adsorption Sciences, 2013, 3, 48-55

http://dx.doi.org/10.4236/jeas.2013.31006 Published Online March 2013 (http://www.scirp.org/journal/jeas)

Microencapsulation of Essential Oils within Alginate:

Formulation and in Vitro Evaluation of
Antifungal Activity
Emad A. Soliman1*, Ahmed Y. El-Moghazy1, Mohammed S. Mohy El-Din1, Magdy A. Massoud2
1
Department of Polymer Materials, Advanced Technology and New Materials Research Institute, City of Scientific Research and
Technology Applications, New Borg El-Arab City, Egypt
2
Department of Plant Protection, Faculty of Agriculture-Saba Basha, Alexandria University, Alexandria, Egypt
Email: *emadsoliman@mucsat.sci.eg

Received January 21, 2013; revised February 22, 2013; accepted March 1, 2013

ABSTRACT
Essential oils (EOs) are the volatile lipophilic components extracted from plants. Many EOs have demonstrated strong
antimicrobial properties when tested in in vitro experiments. The commercial applications of these EOs require a suit-
able formulation constituted by biodegradable compounds that protect them from degradation and evaporation at the
same time that allows for a sustained release. The objective of this study was therefore to reduce the rate of evaporation
of the oil via microencapsulation. Alginate microspheres (AMSs) were prepared using emulsion extrusion method. The
AMSs were hardened with a cross-linking agent, calcium chloride. The effects of the three variables: alginate concen-
tration (0.5% - 8%), the amount of cross-linking agent (0.125% - 2%) and time of cross-linking (5 - 30 min.) on loading
capacity and encapsulation efficiency (EE, %) were studied. The effect of the amount of cross-linker was significant on
loading capacity (%) and EE (%). The AMSs under the optimized conditions provided loading capacity of 22% - 24%
and EE of 90% - 94% based on type of EO. The antifungal activity of vapors of microencapsulated and non-microen-
capsulated oils were evaluated against two of pathogenic fungi species for stored grains: Aspergillus niger and Fusa-
rium verticillioides. The optimized MSs were observed to have a sustained in vitro release profile (50% of the antifun-
gal activity was maintained at the 8th day of the study). In conclusion, encapsulation in Ca-alginate microspheres may
effectively reduce the evaporation rate of essential oils, thus increase the potential antifungal activity.

Keywords: Essential Oils; Alginate Microspheres; Extrusion; Antifungal Activity

1. Introduction vitro evaluation has been conducted on the liquid phase

[2]. However, the biological activity of EOs can be lost
Nowadays, there is a global concern for the widespread
by volatilization of active components or its degradation
use of pesticides which have significant drawbacks in-
by act of high temperatures, oxidation and UV light [4].
cluding increased cost, handling hazards, concern about
These disadvantages make the commercial application of
pesticide residues on food, and threat to human health
these oils limited. On the other side, EOs in the vapor
and environment [1]. Public awareness of these risks has
phase have the potential for use as fumigants [5]. There-
increased interest in finding safer pesticides or alternative
fore, formulation of essential oils involves their prepara-
food protectants to replace synthetic chemical pesticides.
tion in liquid forms (emulsions, micelles, liquid solutions
Therefore, the interest in natural products having an an-
etc.), semi-liquid forms (gels, liposome, etc.) or solid
timicrobial activity to preserve food quantity and quality
forms (microcapsules or microspheres) have to be em-
has increased, because they tend to have low mammalian
ployed for controlling release of active ingredients and
toxicity, less environmental effects and wide public ac-
protecting them from the external environment. Micro-
ceptance [2,3].
encapsulation as one of the most efficient methods of
Many essential oils (EOs), such as garlic, cinnamon,
formulation by which solids, liquids or even gases have
thyme, oregano, clove, basil, coriander, citrus peel, euca-
the desired characteristics in extremely tiny amounts may
lyptus, ginger, rosemary, and peppermint, among others,
be enclosed in microscopic particles formation of thin
have been demonstrated antimicrobial activity.
coatings of wall material with limited permeability
A significant body of research on EOs concerning in
around the substances [6]. There are many researches on
*
Corresponding author. microencapsulation of EOs using several methods in-

Copyright 2013 SciRes. JEAS

 E. A. SOLIMAN ET AL.
cluding spray-drying [7], simple coacervation [8], com-
plex coacervation [9] emulsion extrusion [10] and super-
critical fluid precipitation [11]. Emulsion extrusion is
considered as the most common approach of microencap-
sulation and might be achieved by emulsifying or dis-
persing the hydrophobic components in an aqueous solu-
tion where gelation occur (ionotropic or thermal) [10].
By using emulsion extrusion for microencapsulation, a
broad selection of polymer coatings (shell) and meth-
ods of deposition are available, which are easily adapt-
able to large-scale production. Alginates are natural
commonly used as wall materials since it shows high
toughness and it has considerable effects on the me-
chanical stability of beads. Chemically, alginates are
naturally occurring polycarbohydrates consisting of co-
polymers of -L-glucuronic acid (G) and -D-man-
nuronic acid (M). The relative amounts of these two
building blocks influence the total chemistry of this bio-
polymer, where G/M ratio determines the permeability
properties of the swollen alginate gel which envelops the
essential oils [12].
The main aim of the present study is to develop mi-
croencapsulation of clove, thyme and cinnamon oils in
calcium alginate was done by employing the emulsion
extrusion technology in order to achieve their optimal
antifungal activity against two of pathogenic fungi spe-
cies for stored grains: Aspergillus niger and Fusarium
verticillioides. Various operational parameters such as
concentration of alginate and cross-linking agent and
time of cross-linking on loading capacity and encapsula-
tion efficiency (EE, %) were studied. Furthermore, this
experimental study provides an insight for relating the
antifungal efficiency of microencapsulated essential oils
with storage time.
2. Materials and Methods
2.1. Materials
Three types of essential oils for medical use were pur-
chased from the local market which are extracted from
Clove (Eugenia caryophyllata), thyme (Thymus vulgaris)
and Cinnamon (Cinnamomum zeylanicum), sodium algi-
nate (low viscosity) and n-hexane were purchased from
Sigma-Aldrich, (Germany). Calcium chloride (CaCl2)
was purchased from Park Scientific limited (UK). So-
dium citrate was obtained from Pratap chemical indus-
tries (India). All other reagents were analytical grade and
used without further purification.
2.2. Fungi Species
Two species of stored grains pathogenic fungi, Aspergil-
lus niger and Fusarium verticillioides were obtained
from Genetic Engineering and Biotechnology Research
Copyright 2013 SciRes.
49
Institute (CSRTA). The culture of each fungus were
maintained on Czapeks-Dox agar and stored at 4C.
2.3. Encapsulation of EOs
Micro-encapsulation of oil was conducted using emul-
sion extrusion technique described by Chan [13]. Where,
sodium alginate was dissolved in distilled water to pro-
duce alginate solutions with concentration of 0.5, 1, 2, 4
or 8 w/v%, the solutions were left standing for 24 h to
disengage bubble before use. Afterwards, sodium algi-
nate solution (30 g) and EO (10 g) were homogenized
into a 200 mL beaker with stirring at a speed of 300 rpm
for 45 min by a magnetic stirrer. The oil was gradually
added to the alginate solution during mixing until the
desired oil loading was obtained. Fifty milliliters of algi-
nate-oil emulsion was then sprayed into a collecting wa-
ter bath containing calcium chloride solution (0.125, 0.25,
0.5, 1, 2 w/v%) using an Inotech Encapsulator IER-50
(Switzerland) with a 500-m nozzle [14]. This operation
was adjusted at a frequency of 550 Hz and a voltage of
1.40 kV. The resulting microcapsules were allowed to
harden in the CaCl2 solution for 5, 10, 15, 20, 25 or 30
min. The oil-loaded alginate beads were collected from
the cross-linking solution using a sieve. Finally, the mi-
crobeads were rinsed twice with distilled water, tissue
paper was used to absorb the surface excessive water and
oil onto the wet microcapsules. Quantification of EOs
loaded within AMSs was conducted by using the method
described by Parris and his colleagues [15] with a slight
modification, where the amount of EO enveloped into the
microbeads was quantified by extracting the loaded oil
from 0.5 g of these beads via their dissolution with 5 mL
of sodium citrate (0.055 M) and 5 mL n-hexane. The
absorbance was then measured at wavelength of 275 nm
for thyme oil and at 280 nm for clove and cinnamon oils
by using spectrophotometer model Ultrospec 2000
(Pharmacia Biotech Co. Cambridge, England). The amounts
of EOs were calculated from plotted the standard curves
for EOs with usage of dissolved alginate microbeads
with no EO as a control.
Loading capacity (%) was calculated from the follow-
ing equation:
Loading capacity % Wo WMS 100 (1)
where
Wo = Quantity of loaded EO,
WMS = Quantity of MSs.
Encapsulation efficiency (EE, %) was calculated from
the following equation:
EE % Wo WI 100 (2)
where
Wo = Quantity of loaded EO,
WI = Initial quantity of EO.
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50 E. A. SOLIMAN ET AL.
For each formulation, the loading capacity and encap-
sulation efficiency were determined for triplicate, the
average was calculated.
2.4. Scanning Electron Microscopic Analysis of
MSs
The EOs microspheres were observed visually and ana-
lyzed using high-resolution scanning electron microscope
(SEM) with suitable accelerating voltage and magnifica-
tions. The SEM analysis was carried out at Central
Laboratory of Materials Characterization of Advanced
Technology and New Materials Research Institute
(ATNMRI), City of Scientific Research and Technology
Applications (SRTA) (Egypt) using JOEL 6360LA scan-
ning electron microscope (JEOL Ltd., Tokyo, Japan)
operated at an acceleration voltage of 15 kV. Fine
coat-ion sputter device, JFC1100E (JOEL Ltd., Tokyo,
Japan), consisting of basic and rotary pump unit was
used to perform efficient and rapid metal coating. The
coating pressure was 0.17 torr at 1200 V and 8 mA. The
scanning electron micrographs were used for elucidating
the structural characteristics of alginate microspheres.
2.5. Assessment of Antifungal Activity of Vapors
of Encapsulated EOs
Antifungal activity was assessed by the method described
by Feng and others [16] with a slight modification, these
biological assays were conducted using Petri plates (di-
ameter 90 mm) of Czapeks-Dox agar medium (15 ml/
plate) with making well (diameter 10 mm) in the center
of Petri plates using a flamed cork borer and inoculated
with 100 L suspension of the tested fungi from one-
week old culture (A disc with diameter of 3 cm of each
fungus was cut from periphery and suspended into 10 mL
of sterile distilled water. Then the resulting was vigor-
ously shaken). A sterilized filter paper disc with diameter
of 30 mm was fixed in the upper lid of the Petri dish.
Varying volumes of EO (1, 2.5, 5 and 10 l was placed
in each plate either in free or micro-encapsulated form.
Where, definite volume of free EO was distributed onto
filter paper (at the center). However, the certain amounts
of MSs were adhered using one milliliter of solidification
sterilized agar that was first placed at the center of the
upper lid of the Petri dish. So the volume of EO envel-
oped into each amount of MSs was equivalent to vol-
umes 1, 2.5, 5 and 10 l of EOs. Blank filter paper disc
served as a control for the free oils while for the encap-
sulated oils the control was alginate microbeads (with no
EOs). The surfaces of oil-loaded filter paper discs or ad-
hered MSs ware at a distance of about 6 mm from the
medium surface (the growth surface of the tested fungi).
Plates were tightly sealed with an adhesive tape and in-
cubated at 27C 2C for 5 days. The growth inhibition
Copyright 2013 SciRes.
was determined by measuring the diameter of fungal
growth zones. Three replicates were used per each test
and percentage of growth inhibition was calculated by
the following equation [17]
Growth inhibition % Dc Dt Dc 100 (3)
where
Dc = diameter of fungal colony in control set,
Dt = diameter of fungal colony in treatment set.
2.6. Antifungal Activity Profile of Vapors of
Encapsulated EOs
To assess the antifungal efficiency of free and encapacu-
lated oils during storage, the oil distributed onto filter
paper disc (free oils) and encapsulated oil (MSs) were
stored at 28C 2C for 2, 4 and 8 days. Afterwards, the
biological assay was done for the stored samples as de-
scribed above by using dosage of 5 l/plate against A.
niger.
2.7. Statistical Analysis
Data of the present study were subjected to the analysis
of variance test (ANOVA) as complete randomized de-
sign. The least significant differences (LSD) at the 5%
level of probability were determined using a computer
program [18].
3. Results and Discussion
3.1. Operational Parameter Affecting Loading
Capacity and Encapsulation Efficiency
Influence of various operational parameters of microen-
capsulation process on the loading capacity (%) and en-
capsulation efficiency (EE, %) including alginate con-
centration, cross-linking agent concentration and time
were investigated.
3.2. Alginate Concentration
To explore the effect of alginate concentration on the
loading capacity of the obtained beads, different concen-
trations of alginate (0.5% - 8%) were tried with keeping
the concentration of calcium chloride constant at 0.5%
and cross-linking time of 20 min. The results presented
in Figure 1 which indicated that the loading capacity
increased with increasing the alginate concentration from
0.5% to 2%, where it reached about 23% for thyme,
clove and cinnamon oil. Whereas, further increase in
alginate concentration led to decrease the loading capac-
ity to ~5% with the different oils at alginate concentra-
tion of 8%. This increase in the loading capacity with
increasing the alginate concentration can be attributed to
that increasing of alginate concentration leads to forming
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 E. A. SOLIMAN ET AL.
Figure 1. Effect of alginate concentration on the loading
capacity and the encapsulation efficiency.
a dense network structure with cohesive vacancies (pores)
that entrap the essential oils droplets (proper pores size
with homogeneous distribution). These results are in
agreement with those reported by Manjanna and his
co-workers who found that the encapsulation efficiency
increased with increasing concentration of the sodium
alginate [19]. While, the decline in the loading capacity
with increasing the alginate concentration over 2% can
be explained on the basis that this increases the space
occupied by alginate causing a decrease of the free vol-
ume within the polymer matrix (a compact structure with
smaller pores sizes), and subsequently the amount of oil
that can be entrapped within these pores will be de-
creased. This assumption was supported by findings of
Sevda and Rodrigues that indicated that higher alginate
concentration leads to a decrease of the pores sizes
forming in the resultant microbeads [20].
3.3. Calcium Chloride Concentration
Cross-linking agent concentration as an affecting factor
on the loading capacity was studied by using varying
concentrations of calcium chloride (0.125% - 2%) with
maintaining alginate concentration at 2% and cross-
linking time of 20 min. The results of this study were
presented in Figure 2 indicating that the increase of cal-
cium chloride concentration from 0.125% to 0.5% leads
to increase the loading capacity of the resultant micro-
beads to reach ~23% for thyme, clove and cinnamon oil.
These results are in agreement with those obtained by
Manjanna and his colleagues who found that by increas-
ing calcium chloride concentration from 1% - 5%, the
drug entrapment efficiency were found to increase from
~83% to ~93% [19]. Nevertheless, increasing concentra-
tion of the calcium chloride over 0.5% led to a gradual
decrease in the loading capacity to reach about ~18%,
12%, and ~15% for thyme, clove and cinnamon oil, re-
spectively at calcium chloride concentration of 2%. It is
obvious that increasing calcium chloride concentration
certainly leads to introduce higher levels of Ca2+ ions
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51
Figure 2. Effect of calcium chloride concentration on the
loading capacity and the encapsulation efficiency.
causing cross-linking between the alginate chains and
consequently forms a dense cohesive structure resulting
in more capacity for the resultant beads to entrap higher
amount of oil this supported also by findings of Man-
janna and others [19]. While, decrease of the loading
capacity of the obtained micro-beads with increasing the
calcium chloride concentration over 2% can be explained
on the basis of the higher approaching of the alginate
chains and subsequently decreasing the pores sizes
forming within alginate matrices resulting for squeezing
the gel micro-beads leading to decrease of the loading
capacity.
3.4. Cross-Linking Time
The cross-linking time is also another parameter of
cross-linking process affecting the loading capacity of
oil-encapsulated micro-beads. Therefore, the encapsula-
tion process was done using alginate concentration of 2%
and concentration of calcium chloride of 0.5%, whereas
time of the cross-linking process was changed from 5 to
30 min. Results of these experiments shown in Figure 3
which indicated that extending cross-linking time from 5
to 20 minutes leads to increase the loading capacity to
~23%, ~24% and 22% for thyme, clove and cinnamon oil,
respectively. Notwithstanding, extending the time of
cross-linking over 20 min. leads to decrease the loading
capacity till reach ~18%, ~20% and ~14% for thyme,
clove and cinnamon oil, respectively with cross-linking
time of 30 min. These results can be elucidated on the
basis of gelling (alginate cross-linking) performance, the
cohesion of the formed gel and partition of the oil be-
tween the two environments (within the micro-beads and
aqueous medium) that consequently affect entrapping the
oil within the polymer matrices or its releasing outside
the beads. Where, the released amount of oil is more
pronounced when the time of gel formation extended
[21]. These results supported by findings of Lai and his
co-workers who found that the essential oil encapsulation
efficiency decreased when the cross-linking time in-
JEAS
52 E. A. SOLIMAN ET AL.
Figure 3. Effect of cross-linking time on the loading capac-
ity and the encapsulation efficiency.
creased [22]. Moreover, Kulkarni and his colleagues
found that the percentage of entrapment efficiency de-
creased with increase in the time of exposure to the
cross-linking agent from 10 to 30 min. by about 15%
[23]. This behavior is probably due to a partitioning of
the oil components into the aqueous phase by increasing
the contact.
From the previous results, the best formulation was:
Sodium alginate concentration: 2% w/v, calcium chloride
concentration: 0.5% (w/v) and cross-linking time: 20 min.
Which gave maximum loading capacity (thyme oil:
~23.4%, clove oil ~23.5% and cinnamon oil ~22.5%) and
maximum encapsulation efficiency (thyme oil: ~94%,
clove oil ~94% and cinnamon oil ~90%).
3.5. Microstructure of AMSs
Topographical features and microstructure of plain algi-
nate microspheres (non-loaded with EO) or that having
EO-loaded highest loading capacity (best formulation
prepared at the optimum conditions) were shown in Fig-
ure 4. The micrograph of plain alginate microspheres
(non-loaded with EO) (Figure 4(A)) exhibited that the
surface of these spheres had smooth textural characteris-
tics (high compressive strength, non-shown data). How-
ever, EO-loaded Ca-alginate microspheres (Figure 4(B))
exhibited crimpy surface with appearance of noticeable
lumps. This can be explained on the basis of the deposi-
tion of EO droplets onto the outer or inner surface of the
external layer of alginate microbeads, where the presence
of oil components onto alginate surfaces can lead to plas-
ticize their structure and forming these lumps. On the
other side, cross-section of EO loaded Ca-alginate mi-
crospheres (Figure 4(C)) indicated that the structure of
these beads appear as a three-dimensional, porous sponge.
This cellular structure can be clearly shown in Figure
4(D) that exhibited cavity of the cell and its wall. Fur-
thermore, the wrinkling of cell wall appeared obviously.
This egg-box structure confirms the entrapment of EOs
into the cells of alginate matrix.
Copyright 2013 SciRes.
Figure 4. SEM graphs of alginate microspheres, plain (A),
EO-loaded (B), cross-section of EO-loaded MS (C) and its
microstructure (D).
3.6. Antifungal Activity of Vapors of
Encapsulated EOs
The antifungal activity of the vapors of tested EOs (cin-
namon, clove and thyme) was evaluated with radial
growth technique at several dosages (1, 2.5, 5 and 10 l/
plate) against two species of grains pathogenic fungi.
3.6.1. Aspergillus Niger
Data in Figure 5 clarified that cinnamon oil was the su-
perior oil which; recorded 100% growth inhibition start-
ing at 2.5 l against A. niger comparing with clove and
thyme oils which recorded 83% and 71% growth inhibi-
tion with 2.5 l receptively, while they showed entire
inhibition beginning from dosage of 5 l. These findings
are in agreement with the results reported by Guynot and
others who reported that Aspergillus niger was totally
inhibited by vapor of pure cinnamon, clove and thyme
essential oils at dosage of 50 l [24]. However, Paster
and his colleagues showed that thyme essential oil vapor
produced a significantly reduction in colony diameter of
A. niger at 3 l/L [2]. Whereas, the results showed that
the oils beads had a strong inhibition effect against A.
niger causing complete inhibition begging from 5 l/
plate. While the beads of cinnamon, clove and thyme oils
gave growth inhibition 92%, 87% and 79% at the dosage
of 5 l, respectively.
3.6.2. Fusarium Verticillioides
Results shown in Figure 6 revealed that cinnamon,
thyme and clove oils had higher antifungal activity
against F. verticillioides. Where, cinnamon oil was the
most effective one by giving 100% growth inhibition at 1
l/plate. Thyme oil showed also a high efficiency toward
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 E. A. SOLIMAN ET AL.
Figure 5. Antifungal activity of vapors of the free and en-
capsulated essential oils against A. niger.
Figure 6. Antifungal activity of vapors of the free and en-
capsulated essential oils against F. verticillioides.
this fungus by achieving 100% growth inhibition at dose
of 5 l/plate. However, clove oil gave 100% growth in-
hibition at dosage of 10 l/plate. Soliman and Badeaa
found that the thyme and cinnamon oils had high anti-
fungal activity against Fusarium verticillioides [25].
Whilst, the antifungal activity results of encapsulated oils
indicated that cinnamon oil-loaded alginate beads have
the highest activity. Where, they gave 100% growth in-
hibition for this fungus at the lowest dosage (1 l/plate)
as the free oil. Moreover, the activity of thyme oil beads
were more pronounced than these for clove oil, where
they showed antifungal activity of 65% and 39% respec-
tively, at the lowest applied dosage.
By comparing the antifungal activity of vapors of both
of free and loaded oils as shown in Figures 5 and 6, no
significant difference has been observed at comparable
dosage. This indicated that the efficiency of oils-loaded
in alginate matrix did not affect by the encapsulation
process. These results are in agreement with those ob-
tained by Pandey and his co-workers who found that
formulation of different polymers gel containing carda-
mom, coriander and cinnamon essential oils does not
decrease their antimicrobial activity [26]. Also, Leimann
and others found that the encapsulation process of lem-
ongrass essential oil did not cause any deterioration in
Copyright 2013 SciRes.
53
the essential oil, and their results indicated that there
were no significant alterations in the EO during the mi-
croencapsulation and also that the biological activity of
the essential oil was not affected by the microencapsula-
tion process [27]. On the other side, to explore the capa-
bility for both of free and encapsulated oils to maintain
their antifungal activity through storage, the activity pro-
file of free and loaded oils were detected.
3.7. Antifungal Activity Profile of Encapsulated
EOs
The activity profile of free and encapsulated tested oils
was taken part against A. niger using dosage of 5 l/plate
which gave fully growth inhibition with the encapsulated
and free tested oils after different times (0, 2, 4, 8 days).
The results were shown in Figure 7. They exhibited that
the antifungal activity of free oils was decreased by about
90% after storage periods of 2 days. Whereas, after stor-
age of 8 days as shown in Figure 7, the antifungal activ-
ity of these oils became so weak or almost nil, where
cinnamon oil didnt exhibit any activity. On the other
side, the encapsulated clove and thyme oils still maintain
around half of their antifungal activity even after 8 days
of storage. Whereas, this activity was reduced to 29% in
case of cinnamon oil. These results are in agreement with
findings of Passino and others [28]. These results can be
attributed to volatilize the most of volatile components
responsible for the antimicrobial characteristics of these
oils. Normally, this effect was more pronounced for the
free oils comparing with these encapsulated in alginate
matrix. Whereas, the difference between the antifungal
activities of these three oils after storage depends on the
volatilization extent of their active components.
4. Conclusion
Based on the pervious results, it can be indicated that the
encapsulation process of tested oils can be considered as
inexpensive and efficient technique to maintain the anti-
fungal activity of these oils and promote the ease of han-
Figure 7. Antifungal activity profile of free and encapsu-
lated oils against A. niger.
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54 E. A. SOLIMAN ET AL.
dling of the oils, where these microencapsulated oils,
thyme, clove and cinnamon exhibited high growth inhi-
bition for Aspergillus niger and Fusarium verticillioides.
Moreover, these EO-loaded Ca-alginate microspheres
can maintain 30% - 50% of the antifungal activity of
their enveloped oils after storage time of 8 days based on
the type of these oils, whilst all types of these free EOs
lost their entire antifungal activity after two days of stor-
age. The results obtained from this study suggest that of
all formulation techniques, microencapsulation seems to
be the best choice for increasing the use of EOs. The
microencapsulation in alginate is expected to be suitable
for other hydrophobic essential oils.
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